The emphasis of this paper is upon the sensitivity of the crude leaf extracts of Melia azedarach against some of the hospital isolated human pathogenic bacterial strains. Five plant extracts (Petrol, Benzene, Methanol, Ethyl acetate and Aqueous) under five different concentrations (1mg/ml, 2mg/ml, 5mg/ml, 10mg/ml and 15mg/ml) were tested by the Disk diffusion method. Methanol, Ethyl acetate and Aqueous extracts of the plant showed significant inhibition against bacteria tested. A comparison was made among the sensitivity demonstrated by the leaf extracts and known medicinal and ethnobotanical uses

Khan, Abdul Viqar

Khan, Athar Ali

Shukla, Indu

English

OpenSIUC

Ethnobotanical Leaflets 12: 439-445. 2008.  In Vitro Antibacterial Potential of Melia azedarach Crude Leaf Extracts Against SomeHuman Pathogenic Bacterial Strains Abdul Viqar Khan,1* Athar Ali Khan2 and Indu Shukla3 1, 2 Department of Botany, Faculty of Life Sciences, Aligarh Muslim University Aligarh , 202002, India3 Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh , 202002, India *Corresponding Author: viqarvicky@rediffmail.com Issued 25 June 2008 Abstract          The emphasis of this paper is upon the sensitivity of the crude leaf extracts of Melia  azedarach  against some of thehospital isolated human pathogenic bacterial strains. Five plant extracts (Petrol, Benzene, Methanol, Ethyl acetate andAqueous) under five different concentrations (1mg/ml, 2mg/ml, 5mg/ml, 10mg/ml and 15mg/ml) were tested by the Diskdiffusion method. Methanol, Ethyl acetate and Aqueous extracts of the plant showed significant inhibition against bacteriatested. A comparison was made among the sensitivity demonstrated by the leaf extracts and known medicinal andethnobotanical uses.Key Words: Melia  azedarach, Pathogenic bacteria, inhibition. Introduction          Plant species have been exploited for the treatment of disease by different ethnic societies residing in different partsof the world. But traditional cultures without the knowledge of modern research are not able to trace the importance of plantspecies for human beings and science has identified the unique techniques to test the beneficiary effects of plantcompounds.          According to the World Health Organization (WHO), infectious diseases are the number one cause of deaths worldwide and account for 50 % of the deaths in tropical countries. The number of Multiple Drug Resistant microbial strains orthose with a reduced susceptibility to antibiotics are increasing yearly, and this is attributed to indiscriminate use of broadspectrum antibiotics, surgery, epidermidis of HIV infections etc. In the last few decades, pharmacological industries haveproduced a number of antibiotics, but the resistance of microbes has increased. It has been reported that bacterial strainshave developed resistance to almost all the antibiotics. Further more, some antibiotics have serious undesirable side effectswhich limit their applications, hence, our ultimate goal is to develop antimicrobial agents which are very effective withminimal unwanted side effects.          Reports say that higher plants are the potential source of novel antibiotics. According to WHO, about 80% ofindividuals from developing countries use traditional medicine, therefore such plant species should be investigated in orderto better understand their pharmacological properties, safety and efficacy. Use of plants extracts and their constituents, bothwith known antimicrobial properties, can be of great significance in therapeutic treatment. In the last few years, a number ofstudies have been conducted worldwide to prove such efficacy. Investigations in this field revealed that many plants possessanti microbial properties that are due to compounds produced by the plants, as, for example, phenolic compounds, tanninsand nitrogenous compounds (Cox,1994, Khan & Khan,2003, Khan etal.,2002 ).          In view of the ethnobotanical uses and medicinal properties of Melia azedarach L. [Meliaceae], it was conjecturedthat this plant might possess antibacterial properties; hence, crude leaf extracts were employed in tests designed to measurethe sensitivity of this species against some hospital isolated pathogenic bacterial strains. Common NamesChina berry, Persian lilac, Pride of India, China tree, Pride of China, Umbrella tree, Umbrella China berry, Indian lilac,Bead tree (English); Bakain, Drek, Dek, Pejri, Padrai (India); Bakainu (Nepal); Thamaga (Burma); Mindi (Java); Inia(Hawaii); Alelaila (Puerto Rico); Jacinto (Panama); Aleli (Venezuela); West Indian lilac, Lilac (West Indies);Lilas (Haiti,French); Cinnamumo (Brazil) and Ois rouge (New Celedonia). Scientific name: Melia azedarach Linn.Family: MeliaceaeDescription: The plant is a small-to medium-sized deciduous tree, 5 to 15 m tall and 30 to 60 cm in diameter. It has aspreading, dense and dark green crown. Its bark is dark or reddish brown, smooth, and becoming fissured. The leaves arealternate. Leaflets have short stalks and are thin, hairless, dark green on the upper surface and paler underneath. They emit apungent smell when crushed. Flowers are purple and fragrant. Fruits or berries are yellow, nearly round, smooth, and fleshy.They are as hard as stone, containing 4 to 5 black seeds. Distribution: Melia azedarach Linn. is native to tropical Asia. It is widespread and naturalized in most of the tropics andsubtropical countries. It was introduced and naturalized in the Philippines and now cultivated even in Manila. (Kirtikar &Basu, 1935, Anonymous, 1976, Asolkar etal., 1992). Known medicinal uses:     Leaves:  leprosy,  scrofula,  anthelmintic, antilithic, diuretic, deobstruent, resolvent. Root:  resolvent,deobstruent. Seeds: rheumatism.Leaves: Leaf extract has insecticidal property (azadirachtin) that repels insects in clothing. The leaves can also serve as feedfor goats. Seed oil: The oil is the most active medicinal product of the plant. It is used as antiseptic for sores and ulcers thatshow no tendency to heal. It is also used for rheumatism and skin diseases such as ringworm and scabies. Internally, the oilis useful in malaria fever and leprosy. Ethnomedicinal  uses in  Northern India*Burns:  Fresh leaf extract  is applied  externally.*Gingivitis (Inflamed bleeding gums):  Fresh leaf extractis used as  mouth wash.*Gonorrhea :  Stem bark infusion 30-50 ml is administered orally  twice a  day.*Spicyfood is not  allowed during the course of  treatment.  Piles (Bleeding:  Leaf  extract  5 ml is administered orallythrice a  day.  *Pyrexia:  Leaf  extract  5-10 ml is administered orally  twice a  day  for 7 days.  (*  less knownuses)  (Khan, 2002). Chemical constituents: Bakayanin, quercitrin, rutin, backalactone 6 b-hydroxy-4-stigmastem-3-one and 6 b-hydroxy-4-campesten-3-one, 4, 5-dihydroxy-7-0-a-L-rhamnopyranosyl-(14)-b-D-glucopyranoside, cystine, serine, arginine,glycine, glutamic acid, threonine, methionine, leucine, lycine, and proline. Pharmacology: Powdered dust of fruit insecticidal, crude extract from wood and bark insecticidal, oil antibacterial.Alcoholic extract (50% EtOH) of leaf anthelmentic, oil with unspecified extract central nervous system depressant, mildanalgesic, depression followed by stimulation in animals. Alcoholic extract (50% EtOH) of stem bark anticancerous,antispasmodic, antiviral ( Rastogi & Mehrotra, 1991, 1993, Rastogi, 1998, Satyavati, etal, 1987 ). Materials and MethodsPlant materialAerial plant parts of Melia azedarach L. [Meliaceae], were collected from different localities of Aligarh district, India.Voucher specimen number [AV014, AV206] of the plant were deposited in the Department of Botany, Faculty of LifeSciences, Aligarh Muslim University, Aligarh , 202002, India.                                                          Melia azedarach L. Preparation of extractsCrude  plant extracts;  were  prepared according  to the protocol  described below (Harbone,1973):1) Freshly dried and healthy plant material (leaf) is ground into fine powder in an electric grinder. Powder so obtained isstored in a dessicator.2) Five hundred g plant powder is refluxed with 95% methyl alcohol (MeOH) in a round bottom flask in a water bath for 10hours. Mother liquor (Crude MeOH extract) is filtered out and residual plant material is again refluxed with 95% methylalcohol for 10 hours. The process is repeated four times to obtain maximum yield of MeOH extract. The extract isevaporated to dryness at 35°C under reduced pressure.3) The dried methanolic extract is refluxed with light petrol (60-80°C) for five hours. After filtration, the residualmethanolic extract is again refluxed with petrol for five hours and filtered. This process is repeated five times. Petrol isevaporated under reduced pressure to obtain petrol soluble extract.4) Petrol insoluble fraction of methanolic extract obtained in step 3 is refluxed with benzene for five hours. Thereafter, itwas filtered and refluxed again with benzene for five hours and filtered. The process was repeated five times. Benzene isevaporated under reduced pressure to obtain benzene soluble extract.5) Benzene insoluble fraction obtained in step 4 is refluxed with ethyl acetate for five hours. Thereafter, it is filtered andrefluxed again with ethyl acetate for five hours and filtered. The process is repeated five times. Ethyl acetate is evaporatedunder reduced pressure to obtain ethyl acetate soluble extract.6) Ethyl acetate insoluble fraction obtained in step 5 is refluxed with methyl alcohol (95%) for five hours, filtered and isrepeatedly refluxed for five times with methyl alcohol (Methanol). The methanolic soluble fraction is evaporated underreduced pressure to obtain methanolic extract, while methanol insoluble residue is discarded.Preparation of aqueous extract Shade dried plant material (500 g) is ground to a fine powder, poured with distilled water, and left for 72 hours at roomtemperature.The flask is then refluxed over a hot water bath for 10 hours and the mother liquor is filtered. The solute is again addedwith solvent (distilled water) that is again refluxed and filtered; this process is repeated for 4 times. The filtrate, thusobtained, is evaporated to complete dryness under reduced pressure on a water bath. The residue thus obtained is theaqueous plant extract. Yields per 1000 g dry material: petrol ~ 11 g, Benzene ~ 12.5 g, EtOAC ~ 9.0 g and MeOH ~ 14.0 g. Aqueous extractmaterial (500 g) (yield ~ 48.0 g). Dried plant extract were stored in labeled sterilized screw capped bottles at -200C MicroorganismsThe leaf extracts were tested for possible antibacterial activity in the disk assay using eighteen (18) humanpathogenic bacteria listed in Table No. 1. The bacteria were obtained from the bacterial stock, Department of Microbiology,Jawaharlal Nehru Medical College, Aligarh, India. The bacterial cultures were maintained at 4oC on nutrient agar. Anti microbial assay Agar plates (Mueller & Hinton,1941) are inoculated using a sterile swab dipped into culture inoculums adjusted to 1.5x 108bacterial/ml using 0.5 Farland turbidity standard, the agar is streaked in three directions turning the plates by 600 by eachstreak . All the extracts are sterilized by filtration thorough 0.045 m/m membrane filter. The paper disk (Whatman filterpaper no 1) with, 1mg/ml, 2mg/ml, 5mg/ml,10mg/ml and 15mg/ml plant extracts were dried and placed on the agar surfacewith the help of a sterile forceps. Finally press the sensitivity disc with forceps to make complete contact with the surface ofthe medium. Plates are kept at room temperature for 45 minutes.(Pre diffusion time). Inoculated Petri dishes are incubated at370C over night and at the end of the period, inhibition zones formed on the medium are evaluated in mm (Bauer et al.,1966, Cruickshank, 1968; Colle & Marr, 1989). Experiments were repeated thrice and the mean of the triplicate of theresults is summarized in Table No. 1.Studied activity          Antibacterial activity by disc diffusion method (Bauer et al., 1966, Cruickshank, 1968). Diameters of Petri dish anddisc 9.0 cm and 0.6 cm respectively.ResultsPetrol  leaf extract  of  the plant was found to be  effective against  six of  the gram positive and  nine ofthe gram negative  pathogenic  bacteria.  While  benzene extract  inhibited the growth of  three gram positiveand  nine gram negative  bacteria.  Ethyl acetate,  Methanolic fraction and  Aqueous extract  of  the plant werefound to be  effective against  all the tested  bacterial  strains (Table No.1).DiscussionResults  of  the in vitro  antibacterial  activity brought to light  Interesting facts.  Petrol  fraction showsmaximum inhibition  against  Bacillus  subtilis,  Proteus mirabilis and  Shigella flexneri.  (zone of  inhibition6mm/1mg/ml/disk).  Benzene  extract  inhibited the growth of  eleven tested  bacteria and  the maximuminhibition  zone  was recorded against  Proteus mirabilis and  Shigella flexneri  (zone of  inhibition6mm/1mg/ml/disk).  All  the tested  pathogens showed sensitivity against  the ethyl acetate  fraction and  themost  affected bacteria were  Staphylococcus aureus  and  Bacillus  subtilis  (zone of  inhibition9mm/1mg/ml/disk).  While  methanolic  fraction was also found to be  effective against  all the strains andmaximum inhibition  was recorded for Shigella dysenteriae  and Plesiomonas shigelloides.  (zone ofinhibition  7mm/1mg/ml/disk/each),.  Aqueous leaf extract  showed moderate degree of  sensitivity against  alltested  pathogenic  bacteria.        From the results it is clear  that leaves of  Melia  azedarach  L. are effective in controlling  bacterialinfections  caused by both gram positive and  gram negative  strains. During these investigations it becameclear  that the most  effective crude extract  was ethyl acetate,  which  demonstrated  maximum inhibitionfollowed by Methanolic fraction that inhibited the growth of  all the tested  human pathogens. The petrol  andbenzene extracts, as  compared  to the other three extracts, showed anti microbial  action  against  fifteen andtwelve pathogens, respectively.  The phytochemical  screening of  this  plant gave  positive test  for Phenoliccompounds (Harbone,  1973).  It was also noticed  that Methanolic, Ethyl acetate  and  Aqueous extractsshowed antibacterial  activity against  all of  the pathogens (Table No.1). The above  results show that plantextracts can  be  effective antibiotics, both in controlling  gram positive and  gram negative  human pathogens.The in vitro  screening also confirms medicinal  uses reported  earlier. (Kirtikar & Basu, 1935, Anonymous,1976, Asolker etal.,  1992, Khan, 2002, Jain 1991, Fransworth,1988, Khan etal.,2002).AcknowledgementThanks are due to Department of Science and Technology SERC Division, New Delhi for financial support to the author DrAbdul Viqar Khan (Project Title: Anti-bacterial Screening of some Ethnomedicinally important plants against some Humanpathogenic Bacteria DST No: 304/2004:SR/FT/L-129/2004)BIBLIOGRAPHYAnonymous  (1976) Indian material  medica,  Vol.1,  pp.283-284, Population Prakashan, New Delhi,  India. Asolker LV,  Kakkar  KK & Chakra  OJ. (1992) Second  supplement to glossary  of  Indian medicinal  plants  withactive  principles, part 1(A-K). XIVII+414,  Pub. & Inf. Div.(CSIR),  New Delhi. Bauer  AW, Kirby WMM & Sherries  T. (1996) Antibiotic susceptibility  testing by a  standard  single  discmethod. American  Journal of  clinical pathology 45,493. Colle JA & Marr W.  (1989) Cultivation of  Bacteria . In  Mackie & Mc Cartney:  Practical microbiology13th ed .  pp.121-140, Churchill  livingston,  USA. Cox PA. (1994) The ethnobotanical approach to drug  discovery: strengths and limitations . In,  ethnobotanyand  the search for the new drugs.  pp. 25-36, John  Wiley  & Sons.  England. Cruickshank R. (1968) Medical  Microbiology  :  A guide to diagnosis  and  control  of  infection, pp.888,Edinburgh and  London :  E & S Livingston Ltd. Fransworth NR.  (1988) Sereening plants  for new medicines . Wilson  EO(ed.).  Biodiversity,  pp.83-97,National  Academy Press, Washington. Jain SK. (1991) Dictionary  of  Indian Folk  medicine and ethnobotany,  pp.XII+311,  Deep publication,N.Delhi. Khan A.V.  & Khan A.A.  (2003) herbal abortifacients  used by folk people of  some districts of  Western  UttarPradesh (India).  Journal of  Natural Remedies  3(1),  41-  44. Khan AV,  Parveen G,.  Alam MM & Singh VK.  (2002) Ethnomedicinal  uses of  Neem in rural  areas of  UttarPradesh, India. Ethnomed & Pharmacog . II Rec. Prog. In  Med.  Plants  pp. 7, 319-326, (Sci. Tech. Pub.USA). Khan AV,  Alam MM & Singh VK.  (2002). Ethnomedicinal  uses of  Citrullus colocynthis  (L.)  Schard.  In  ruralareas of  Aligarh District of  Uttar Pradesh, India Ethnomed. & Pharmacog. II.  Rec. Prog in Med.Plants. 7, 383-388, (Sci. Tech. Pub. USA). Khan AV.  (2002) Ethnobotanical studies on plants  with medicinal  and anti-bacterial properties  pp. 1-293.(Thesis).  Aligarh Muslim University,  Aligarh. Kirtikar KR & Basu BD.  (1935) Indian Medicinal Plants . Vol 3, pp. 1841. Allahabad. Lalit  MohanPublication. Mueller JH & Hinton J.  (1941) A protein  free medium for primary isolation  of  the gonococcus andmeningococcus.  Procedings of  society  of  exp  Biology and Medicine  48, 330-333. Rastogi RP & Mehrotra BN.  (1991) Compendium of  Indian Medicinal Plants , Vol.I,1:833 CSIR Publication,India. Rastogi RP & Mehrotra BN.  (1993)  Compendium of  Indian Medicinal Plants , Vol. III,1:  831 CSIRPublication, India. Rastogi RP .(1998) Compendium of  Indian Medicinal Plants , Vol. V  1:1060 CSIR Publication, India. Satyavati.  GV,  Gupta  AK & Tanabu N. (1987) Medicinal plants  of  India Vol.2 pp. XI+557, CSIRPublication, Indian Council of  Medical  Research, Cambridge  printing worker,  N. Delhi. Harbone  J  B. (1973) Phytochemical  Methods. A guide to Modern Technique of  Plant Analysis, pp X+277,Chapman and  Hall  ,London, Topman and  Hall  ltd, Tokyo, Japan  Table 1. In vitro antibacterial potential of Melia azedarach crude leaf extracts against some human pathogenic bacterialstrains Inhibition zone (mm) Gram Positive Bacteria Gram Negative Bacteria 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11PETROL1 5 - - 5 5 - 6 - 4 - 6 3 3 4 4 - 6 22 8 4 4 10 8 - 10 4 6 - 10 5 6 7 7 - 10 55 10 7 7 16 11 - 13 7 12 - 14 9 11 11 11 - 14 1110 16 11 12 17 16 - 19 9 17 - 17 11 12 15 15 - 19 1415 21 18 17 22 18 - 23 13 22 - 21 13 17 19 20 - 22 17BENZENE1 5 - - - 5 - 4 4 4 - 6 3 3 4 4 - 6 52 8 - - - 8 - 7 6 6 - 10 5 6 7 7 - 10 115 10 - - - 11 - 10 11 11 - 14 9 11 11 11 - 14 1410 16 - - - 16 - 15 14 16 - 17 11 12 15 15 - 19 1815 21 - - - 18 - 19 19 21 - 21 13 17 19 20 - 22 21ETHYL ACETATE1 9 5 6 6 6 7 9 6 7 6 7 7 9 6 6 7 9 72 12 7 11 10 9 10 12 10 11 11 12 12 12 12 9 10 13 115 19 11 17 16 16 15 17 13 17 16 17 17 18 17 16 17 18 1910 22 16 19 19 20 18 20 17 19 18 22 22 23 19 20 22 22 2315 26 21 23 23 26 23 26 20 23 23 25 27 26 24 24 27 26 27METHANOL1 5 - 4 6 5 5 5 4 3 2 5 6 5 4 5 7 4 72 7 4 6 10 9 9 7 7 5 5 9 10 7 7 8 10 7 115 12 6 11 16 16 15 12 9 8 9 14 15 10 12 12 16 12 1610 16 10 15 19 20 19 17 13 13 14 18 19 13 16 18 20 18 1915 21 16 20 23 26 21 22 17 17 18 22 23 19 21 21 23 22 21AQUEOUS1 4 - 4 6 5 2 4 3 5 2 2 3 2 2 4 2 4 42 7 2 7 10 9 6 7 5 9 5 5 6 5 4 6 5 7 75 10 6 11 12 13 9 10 9 11 8 9 11 8 7 11 10 12 1110 15 8 15 16 18 11 15 11 14 12 14 15 11 12 14 12 15 1615 18 11 19 19 20 16 19 13 16 13 18 19 15 17 19 16 20 19CHLORAMPHENICOL 18 18 16 - - - 16 18 16 - 16 18 - 16 17 19 18 20 Gram Positive Bacteria1.Staphylococcus aureus*  2.Staphylococcus aureus  ATCC 25923  3.Staphylococcus epidermidis* ,  4.  Group - A  Streptococcus*  5.Group - B Streptococcus*  6.  Enterococcus faecalis*  7.  Bacillus  subtilis*.Gram Negative Bacteria1.                           Escherichia coli ATCC 25922. Edwardsiella tarda* 3. Klebsiella pneumoniae* 4. Proteus mirabilis * 5. Proteus vulgaris* 6. Pseudomonasaeruginosa* 7. Salmonella typhi *8. Shigella boydi*i 9. Shigella dysenteriae* 10. Shigella flexneri* 11. Plesiomonas shigelloides*. ATCC American type culture collection,* Hospital isolated pathogenic strainsCHLORAMPHENICOL 10mg/disk,aValues are the mean of replication of three; -, no inhibition.      

\u3cem\u3eIn Vitro\u3c/em\u3e Antibacterial Potential of \u3cem\u3eMelia azedarach\u3c/em\u3e Crude Leaf Extracts Against Some Human Pathogenic Bacterial Strains

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\u3cem\u3eIn Vitro\u3c/em\u3e Antibacterial Potential of \u3cem\u3eMelia azedarach\u3c/em\u3e Crude Leaf Extracts Against Some Human Pathogenic Bacterial Strains

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